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Objective Surfactant dysfunction is an important pathologic disturbance in various forms of acute inflammatory lung injury. Previously we reported the presence of marked alterations in the composition and activity of pulmonary surfactant in bilateral lung contusions (LC) injury induced by blunt trauma in rats. This is extended here to a mouse model of unilateral LC with a focus on compositional and functional changes in surfactant associated with permeability injury and increases in activity of secretory phospholipase A 2 . Results Surfactant-associated gene expression was not altered in mice with unilateral LC injury on the basis of Affymetrix analysis. LC mice had significant permeability injury with increased albumin and total protein in bronchoalveolar lavage at 5, 24, 48, and 72 hours after insult compared with uninjured controls. The percent content of large surfactant aggregates was depleted at all postinjury times, and pulmonary pressure-volume (P-V) mechanics and compliance were abnormal during this period. Surfactant dysfunction was evaluated in 24 hours, when permeability injury and P-V changes were most prominent. At this time, activity levels of secretory phospholipase A 2 were increased in bronchoalveolar lavage, and chromatographic analysis showed that large surfactant aggregates had decreased levels of phosphatidylcholine and increased levels of lyso-phosphatidylcholine. These changes were accompanied by severe detriments in large aggregate surface activity by pulsating bubble surfactometry. Large aggregates from LC mice at 24 hours had minimum surface tensions of only 12.6 ± 1.1 mN/m after prolonged bubble pulsation (20 min) compared with 0.7 ± 0.03 mN/m for uninjured controls. Conclusion These results document important detriments in the composition and activity of pulmonary surfactant in LC injury in mice and suggest that active synthetic phospholipase-resistant exogenous surfactants may have utility in treating surfactant dysfunction in this clinically important condition.

This article examines exogenous lung surfactant replacement therapy and its utility in mitigating clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). Biophysical research has documented that lung surfactant dysfunction can be reversed or mitigated by increasing surfactant concentration, and multiple studies in animals with ALI/ARDS have shown that respiratory function and pulmonary mechanics in vivo can be improved by exogenous surfactant administration. Exogenous surfactant therapy is a routine intervention in neonatal intensive care, and is life-saving in preventing or treating the neonatal respiratory distress syndrome (NRDS) in premature infants. In applications relevant for lung injury-related respiratory failure and ALI/ARDS, surfactant therapy has been shown to be beneficial in term infants with pneumonia and meconium aspiration lung injury, and in children up to age 21 with direct pulmonary forms of ALI/ARDS. However, extension of exogenous surfactant therapy to adults with respiratory failure and clinical ALI/ARDS remains a challenge. Coverage here reviews clinical studies of surfactant therapy in pediatric and adult patients with ALI/ARDS, particularly focusing on its potential advantages in patients with direct pulmonary forms of these syndromes. Also discussed is the rationale for mechanism-based therapies utilizing exogenous surfactant in combination with agents targeting other aspects of the multifaceted pathophysiology of inflammatory lung injury. Additional factors affecting the efficacy of exogenous surfactant therapy in ALI/ARDS are also described, including the difficulty of effectively delivering surfactants to injured lungs and the existence of activity differences between clinical surfactant drugs.

Introduction Lung contusion (LC) from blunt thoracic trauma is a clinically-prevalent condition that can progress to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Patients with LC are at risk for gastric aspiration at the time of trauma, but the combined insults have not been well-studied in animal models. This study tests the hypothesis that concurrent gastric aspiration (combined acid and small gastric particles, CASP) at the time of trauma significantly increases permeability injury and inflammation compared with LC alone, and also modifies the inflammatory response to include distinct features compared with the aspiration component of injury. Materials and Methods Four groups of adult male Long-Evans rats were studied (LC, CASP, LC + CASP, uninjured controls). LC was induced in anesthetized rats at a fixed impact energy of 2.0 J, and CASP (1.2 mL/kg body weight, 40 mg particles/mL, pH = 1.25) was instilled through an endotracheal tube. Lung injury and inflammation were assessed by arterial blood gases and levels of albumin, cells, and cytokines/chemokines in bronchoalveolar lavage (BAL) at 5 and 24 h. Results Rats with LC + CASP had lower mean PaO2/FiO2 ratios compared with LC alone at 24 h, and higher BAL albumin concentrations compared with either LC or CASP alone. Rats with LC + CASP versus LC had more severe inflammation based on higher levels of PMN in BAL at 5 h, increased whole lung myeloperoxidase (MPO) activity at 5 and 24 h, and increased levels of inflammatory mediators in BAL (TNFα, IL-1β, and MCP-1 at 5 and 24 h; IL-10, MIP-2, and CINC-1 at 5 h). Rats with LC + CASP also had distinct aspects of inflammation compared with CASP alone, i.e., significantly higher levels of IL-10 (5 and 24 h), IL-1β (24 h), CINC-1 (24 h), and MCP-1 (24 h), and significantly lower levels of MPO (5 h), MIP-2 (5 h), and CINC-1 (5 h). Conclusions Concurrent gastric aspiration can exacerbate permeability lung injury and inflammation associated with LC, and also generates a modified inflammatory response compared with aspiration alone. Unwitnessed gastric aspiration has the potential to contribute to more severe forms of LC injury associated with progression to ALI/ARDS and pneumonia in patients with thoracic trauma.

This article reviews exogenous surfactant therapy and its use in mitigating acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) in infants, children, and adults. Biophysical and animal research documenting surfactant dysfunction in ALI/ARDS is described, and the scientific rationale for treatment with exogenous surfactant is discussed. Major emphasis is on reviewing clinical studies of surfactant therapy in pediatric and adult patients with ALI/ARDS. Particular advantages from surfactant therapy in direct pulmonary forms of these syndromes are described. Also discussed are additional factors affecting the efficacy of exogenous surfactants in ALI/ARDS, including the multifaceted pathology of inflammatory lung injury, the effectiveness of surfactant delivery in injured lungs, and composition-based activity differences among clinical exogenous surfactant preparations.

The synthesis of three phosphoglycerols is described, one of which contains the previously unknown phosphonoglycerol headgroup. The surface tension-lowering capabilities of synthetic lung surfactant mixtures containing the PG analogs were measured on the pulsating bubble surfactometer and compared to known controls. The PG-containing mixtures exhibited superior surface tension-lowering properties indicating the significant potential of these analogs as components in synthetic exogenous lung surfactants.

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO(2)-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO(2)-lipid and DEPN-8 reached maximum surface pressures of 72mN/m (minimum surface tensions of

Epifluorescence microscopy was used to study the structure and phase behavior of phospholipid films containing a human-sequence monomeric SP-B1–25 synthetic peptide (mSP-B1–25). Measurements were done directly at the air–water (A/W) interface on films in a Langmuir–Whilhelmy balance coupled to a fluorescence microscope and real-time detection system to yield an approximate optical resolution of 1 Am. Fluorescence was achieved by laser excitation of 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3dodecanoyl)-1-hexadecanoyl-sn-glycero-3-PC (BODIPY-PC, concentration V1 mol%). The presence of mSP-B1–25 in films of 4:1 (mol/mol) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) had a substantial effect on lipid morphology and phase behavior that depended on both surface pressure and peptide concentration (10, 5, and 1 wt.%). The mSP-B1–25 peptide tended to fluidize phospholipid monolayers based on expanded molecular areas and reduced collapse pressures. In addition, epifluorescence measurements revealed the formation of solid-phase domains apparent as three-armed counterclockwise spirals separated from regions of fluid liquid-expanded phase domains in compressed phospholipid–peptide films. The appearance of these separated solid-phase domains resembled pure L-DPPC rather than the ensemble-type solid domains found in films of DPPC/DOPG alone and were most apparent when 10 wt.% mSP-B1–25 was present. In contrast, films containing lower, more physiological mSP-B1–25 contents of 5 and 1 wt.% exhibited a prominent intermediate ddendriticT phase that increased in extent as surface pressure was raised. This phase was characterized by branching structures that formed a lattice-like mesh network with fluorescence intensities between a dye-depleted solid domain and a dye-enriched liquid phase. These results indicate that mSP-B1–25 at near-physiological levels produces morphological changes in phospholipid monolayers analogous to those observed for native SP-B1–79. D 2004 Elsevier B.V. All rights reserved.

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d62:DPPG, and 7:1 DPPC-d62:DOPG, alone and in the presence of 0.5–10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH2 antisymmetric and symmetric stretching bands (∼2920 and 2852 cm−1) along with the analogous CD2 stretching bands (∼2194 and 2089 cm−1) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1–2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH2 and CD2 stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d62:DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d62:DOPG monolayers, but in this case an increase in CH2 band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD2 band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d62 chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d62:DPPG. SP-B/C had no significant effect on DPPC-d62 structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.

The bulk shear viscosities of aqueous dispersions of lavaged calf lung surfactant (LS) and its chloroform:methanol extract (CLSE) were measured as a function of concentration, shear rate and temperature. At 10-mg phospholipid per milliliter, dispersions of LS and vortexed CLSE in 0.15 M NaCl (saline) had low viscosities near 1 cp over a range of shear rates from 225 to 1125 s(-1). Lung surfactant viscosity increased with phospholipid concentration and became strongly non-Newtonian with higher values at low shear rates. At 37 degrees C and 40 mg/ml, LS and vortexed CLSE in saline had viscosities of 38 and 34 cp (77 s(-1)) and 12 and 7 cp (770 s(-1)), respectively. Viscosity values for LS and CLSE were dependent on temperature and, at fixed shear, were lower at 23 degrees C than at 37 or 10 degrees C. Hysteresis was also present in viscosity measurements depending on whether shear rate was successively increased or decreased during study. Addition of 5 mM Ca(2+) at 37 degrees C markedly reduced CLSE viscosity at all shear rates and decreased LS viscosity at low shear rates. Dispersion by sonication rather than vortexing increased the viscosity of CLSE at fixed shear, while synthetic phospholipids dispersed by either method had low, relatively Newtonian viscosities. The complex viscous behavior of dispersions of LS and CLSE in saline results from their heterogeneous aggregated microstructure of phospholipids and apoproteins. Viscosity is influenced not only by the aggregate surface area under shear, but also by phospholipid-apoprotein interactions and aggregate structure/deformability. Similar complexities likely affect the viscosities of biologically-derived exogenous surfactant preparations administered to patients in clinical surfactant therapy.

This work compares two reaction schemes for preparing 2,3-bis(fattyalkylthio)-1-propanols for further synthetic adaptation as hydrophobic analogs of lung surfactant phosphatidylcholines. An attempt to prepare 2,3-bis(fattyalkylthio)-1-propanols based on the previously published methods of Bell and co-workers (B.R. Ganong, C.R. Loomis, Y.A. Hannun, R.M. Bell, 1986. Proc. Natl. Acad. Sci. USA 83, 1184-1188; B.R. Ganong, R.M. Bell, 1987. Methods Enzymol. 141, 313-320; J.P. Walsh, L. Fahrner, R.M. Bell, 1990. J. Biol. Chem. 265, 4374-4381) was found to give the rearranged 1,3-bis(fattyalkylthio)-2-propanols as major products. As a reliable alternative, the reaction of ethyl 2,3-dibromopropionate with 2 equivalents of long chain sodium n-alkanethioates gave the corresponding ethyl 2,3-bis(n-alkylthio)propionates, which were then reduced with LiAlH4 to yield the desired 2,3-bis(fattyalkylthio)-1-propanols. Both 13C and 1H NMR spectroscopy were used to differentiate the two possible 1,3- and 2,3-dithio substituted alcohol products and to rigorously assign their structures.

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.

This study investigates the importance of thioester-linked acyl groups in lung surfactant protein C (SP-C) in facilitating interactions with phospholipids that yield functionally important surface active behaviors. Native SP-C, palmitoylated at cysteine residues at positions 5 and 6, was isolated from bovine lung surfactant by liquid chromatography. Deacylated SP-C (dSP-C), unchanged in composition and sequence from SP-C but having a decreased alpha-helical content in films with dipalmitoyl phosphatidylcholine (DPPC) of 52 versus 70%, was obtained by treatment with 0.1 M sodium carbonate buffer at pH 10. Surface activity was studied for SP-C and dSP-C combined with column-purified phospholipids (PPL) from calf lung surfactant or with synthetic phospholipids (DPPC or a synthetic phospholipid mixture (SPL) containing 50:35:15, DPPC:egg phosphatidylcholine:egg phosphatidylglycerol). Interfacial measurements included surface pressure time adsorption isotherms for dispersed surfactants with diffusion minimized, dynamic surface pressure area isotherms and respreading for films in the Wilhelmy balance, and overall surface tension lowering at physiologic cycling rate in oscillating bubble experiments. Dispersions of PPL:SP-C and SPL:SP-C rapidly adsorbed to high equilibrium surface pressures of 47-48 mN/m, significantly better than corresponding dispersions containing dSP-C. The adsorption of PPL:dSP-C was essentially unchanged from that of PPL alone, and the adsorption of SPL:dSP-C was improved only slightly over SPL alone. In Wilhelmy balance studies, dynamic respreading was significantly improved over phospholipids alone in films of SP-C plus PPL, SPL, or DPPC. Respreading was improved less markedly by dSP-C in corresponding films with SPL or DPPC and not at all in films with PPL. Maximum surface pressures were also higher in cycled films of SP-C versus dSP-C combined with PPL or SPL. In bubble experiments (37 degrees C, 20 cycles/min), dispersions of PPL:SP-C and SPL:SP-C reached low minimum surface tensions of1 and 5 mN/m, respectively, whereas PPL:dSP-C and SPL:dSP-C only reached minima of approximately 20 mN/m as did PPL and SPL alone. Acylation in SP-C is crucial for its interactions with phospholipids over the full spectrum of adsorption and dynamic surface behaviors important for lung surfactant.

Aqueous dispersions of dipalmitoyl phosphatidylcholine (DPPC), calf lung surfactant (LS) and an organic solvent extract of calf surfactant (CLSE) were examined by Fourier transform infrared (FTIR) spectroscopy in the presence and absence of calcium. DPPC, the single most abundant constituent of lung surfactant, had FTIR spectra that were essentially equivalent in 0.15 M NaCl with or without calcium (5 m M CaCl 2 or 5 m M EDTA at pH ∼ 5.6). DPPC dispersions had spectral characteristics very similar to solid DPPC, with differences reflecting largely phosphate group hydrogen bonding in the aqueous phase. In contrast, dispersions of CLSE and whole LS had spectral behavior that was calcium-dependent. In the absence of calcium, acyl wagging bands (∼ 1170-1300 cm-1) were significantly decreased in intensity for CLSE compared to DPPC, indicating that the hydrophobic surfactant proteins and secondary surfactant lipids led to fewer phospholipid chains in the trans configuration. Wagging bands were also decreased in LS without calcium compared to DPPC. Phospholipid order was increased in dispersions of both CLSE and LS in 5 m M CaCl 2 compared to 5 m M EDTA. Acyl wagging bands became more prominent for both CLSE and whole LS with calcium, although they were still less intense than for DPPC alone. Acyl stretching bands in the region 2800-3000 cm -1 were broadened and lowered in frequency for CLSE in 5 m M CaCl 2 compared to 5 m M EDTA, also consistent with increased chain order. Additional calcium-dependent SP-A mediated ordering was indicated by even broader symmetrical and asymmetrical acyl stretching bands for whole LS in 5 m M CaCl 2 . Broadening of phosphate asymmetrical and symmetrical stretching bands (∼ 1220 and 1080/1065 cm -1 , respectively) for CLSE and LS dispersions in 5 m M CaCl 2 suggested that calcium also interacted with at least a subset of surfactant phospholipids in the headgroup region. The observed effects on molecular order from the surfactant apoproteins and secondary lipids in LS and CLSE dispersions may be related to their actions in facilitating the adsorption of DPPC to the air-water interface.

Thermal and lyotropic phase behavior was studied by X-ray diffraction and differential scanning calorimetry for a diether phosphonolipid analog (DEPN-8) of the major lung surfactant glycerophospholipid dipalmitoyl phosphatidylcholine (DPPC). DEPN-8 differs in an ether, rather than an ester, bond at the acyl chain-backbone linkage and a headgroup phosphonate (isosteric methylene substitution) versus phosphate constituent. Analysis of lamellar diffraction maxima demonstrated that at high relative humidity (98%) and temperatures below the liquid crystal phase transition (approximately 45 degrees C), DEPN-8 formed interdigitated bilayers with a characteristic periodicity of 41.9-46.5 A. At low humidity the gel phase DEPN-8 bilayers were characteristic of a normal L beta phase with a periodicity equivalent to DPPC (57-59 A). Above the liquid crystal thermal phase transition, bilayer spacing for both DEPN-8 and DPPC was 51-52 A, characteristic of the L alpha phase. Complete assessments of both lamellar and in-plane X-ray scattering used to construct electron density profiles and structure-factor plots for DEPN-8 defined more fully the interdigitated bilayer state at high humidity and low temperature. Compared to DPPC, it is energetically favorable for DEPN-8 to form interdigitated bilayers under conditions of excess water and low temperature. The flexible character of the ether bonds in DEPN-8 allows increased hydrophobic interactions between acyl chains, without generating a steric penalty from the increased packing density of the molecules. Additionally, the ether bond and the phosphonate moiety may allow for more energetically favorable interactions between the choline portion of the headgroup and water. The DEPN-8 ether linkage may also contribute to the improved adsorption and film respreading found previously for this phosphonolipid compared to DPPC.

Dynamic surface pressure-area (π-A) isotherms were measured for dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), and 13 chain-equivalent synthetic phospholipid and phosphonolipid diether and etheramide analog compounds structurally related to these glycerophospholipids. Spread interfacial films at a high initial concentration of 15 A2/molecule were studied under continuous cycling to emphasize maximum surface pressure and dynamic respreading behaviors relevant for pulmonary surfactant. Wilhelmy balance subphases containing NaCl and total ionic strength ∼0.15 were used at pH 2.6, 5.6, and 11.5 to give different headgroup ionization states without significantly affecting compound steric bulk. Intracompound changes in isotherm behavior with pH were correlated with variations in headgroup hydrogen bonding and charge for four groups of molecules: (1) DPPC and analog compounds with choline or similar headgroups (Group I) had π-A isotherms with low minimum surface tensions 70 mN/m) at all three pH values, with dynamic respreading on successive cycles that was lowest at pH 2.6 and increased at pH 5.6 and 11.5; (2) DPPE and related compounds with primary amine headgroups (Group II) had higher minimum surface tensions ranging from 5 to 20 mN/m, with respreading that was greater than that of DPPC-related compounds at pH 2.6 and 5.6, and which decreased dramatically at pH 11.5; (3) secondary amine/ammonium compounds with one N-substitution (Group IIIa) behaved similarly to DPPE and its analogs, with higher minimum surface tensions than DPPC-related molecules and respreading that was maximal at low pH and decreased substantially at pH 11.5; and (4) tertiary amine/ammonium compounds with two N-substitutions (Group IIIb) respread better at pH 11.5 than at 5.6 and 2.6, with strikingly altered isotherms and elevated minimum surface tensions (∼30 mN/m) at high pH. The dynamic π-A isotherm characteristics of the majority of phospholipids and phosphonolipids indicated that increased intermolecular hydrogen bonding between headgroups led to increased dynamic respreading in interfacial films. Additionally, in the absence of substantial intermolecular hydrogen bonding, a cationic lipid headgroup charge (neutral phosphate/phosphonate group) correlated with decreased respreading facility for the surfactant molecules studied.

Dynamic respreading and maximum surface pressures are analyzed as a function of chain-backbone linkage, phosphatephosphonate group, and N-headgroup structure for surface-excess films of dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), and 13 related phospholipid and phosphonolipid analogs. Film behavior varied significantly with molecular structure, related in part to differences in molecular packing and mobility from ether vs amide vs ester linkages, as well as structure-induced variations in headgroup charge, hydration, hydrophobicity, hydrogen bonding, and steric bulk. Analysis of surface pressure-area isotherms from Wilhelmy balance experiments at three different pH values (H. Liu et al., J. Colloid Interface Sci. 167, 378-390) showed that: (1) diether phosphonolipid (DEPN) analog compounds had better respreading on successive cycles into the collapse regime compared to ether-amide phosphonolipid (EAPN) analogs with equivalent N-headgroups at pH 2.6, 5.6, and 11.5; (2) diether phospholipids (DHPC and DHPE) also had better respreading than corresponding diester phospholipids (DPPC and DPPE) at all three pH values; (3) the headgroup phosphonate vs phosphate linkage led to decreased cycle 2/cycle 1 respreading at all pH in the ethanolamine diether phosphonolipid DEPN-13 vs DHPE and in the choline diether phosphonolipid DEPN-8 vs DHPC, although cycle 7/cycle 1 respreading was improved in DEPN-8; (4) maximum surface pressures generated by PC-related compounds (DPPC, DHPC, DEPN-8, EAPN-9) were > 70 mN/m independent of pH, while maximum pressures for PE-related compounds (DPPE, DHPE, DEPN-13, EAPN-13) were lower at 51-67 mN/m and varied more with pH; (5) respreading and maximum surface pressures did not vary in a consistent pattern as a function of pH for DEPN and EAPN analogs with different degrees of N-methylation, particularly at pH 11.5 where DEPN-10 and EAPN-110 with two N -methyl groups paradoxically had the lowest maximum surface pressures and best respreading of any compounds studied; and (6) the N-substitution of two hydroxyethyl groups for two methyl groups had less effect on surface behavior than N-substitution of methyl groups for hydrogen. The significant differences in dynamic respreading and maximum surface pressure found here for relatively small changes in molecular structure and headgroup charge indicate that specific compositional requirements likely exist for glycerophospholipids in functional pulmonary surfactant. The results also support the feasibility of synthesizing phospholipid-like molecules with specific surface-active properties for potential use in new exogenous lung surfactant mixtures.

A series of synthetic analogs of naturally occurring glycerophospholipids has been studied for gel-to-liquid transition temperature (Tc) by using differential scanning calorimetry. The compounds investigated were dihexadecyl phosphatidylcholine (DHPC) plus 11 newly synthesized phosphonolipids. Six were racemates and have ether-ether fatty chains and 5 were chiral (R) with ether-amide fatty chains bonded to the glycerol moiety; each series also contained a methylene isosteric substitution for one of the oxygen atoms in naturally occurring glycerophospholipid phosphate esters. All analogs have fatty chains of equivalent length in saturated carbon atoms C2C16, but varied in N-headgroup structures. The thermal behaviors of these novel molecules were found to depend upon the degree of alkylation of the headgroups and upon whether the hydrocarbon chains are bonded as ethers or amides. The ether-ether (diether) phosphonolipid (DEPN) analogs of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) had increased gel-to-liquid crystal transition temperatures (Tc) compared to their naturally occurring (fatty acyl ester) glycerophospholipid counterparts. In general, DEPN analogs had Tc values 3.7−5.8°C higher than ether-amide phosophonolipid (EAPN) compounds of equivalent headgroup, although EAPN-13 was an exception with the highest Tc of any compound studied. In both the DEPN and EAPN series, analogs with hydrogen atom(s) covalently bonded to the headgroup nitrogen atom showed higher Tc values, possibly related to their increased potential for hydrogen bonding. Interfacial property studies (adsorption, Wilhelmy balance, oscillating bubble) showed that several analogs with higher transitions than DPPC, the principal lung surfactant glycerophospholipid, had improved adsorption and respreading, with equivalent surface tension lowering in compressed spread films on the balance and with minimum surface tensions

A homologous series of chiral (R) ether-amide phosphonolipid analogs of naturally occurring (R) glycerophospholipids were synthesized and characterized for their interfacial behaviors. The phosphonolipids possess isoteric ether, amide, and phosphonate functions at positions corresponding to the sn-1, sn-2, and sn-3 ester functions, respectively, of naturally occurring glycerophospholipids. All compounds were synthesized with disaturated C16:0 alkyl/acyl moieties to give structural analogy with dipalmitoyl phosphatidylcholine (DPPC), the major glycerophospholipid component of lung surfactant. Further substitutions at the headgroup nitrogen were also used to generate differences in headgroup size and polarity in the synthetic compounds. The surface activity of the ether-amide phospholipids was investigated in terms of adsorption to the air-water interface, together with studies of dynamic respreading after monolayer collapse and surface tension lowering in dynamically compressed spread films and dispersions. Results showed that several ether-amide phosphonolipids had more rapid adsorption and improved dynamic respreading behavior compared to DPPC, plus the ability to lower surface tension into the range of less than 1 to 4 mN/m in spread films and in dispersions under dynamic conditions. In combination with a series of diether phosphonolipids synthetized in a companion study [1], these ether-amide compounds are useful in the development of molecular structure-surface activity correlates for lung surfactant-related materials, and should assist in investigating the specificity of interactions between phospholipids and other pulmonary biological molecules.

The effects of surfactant protein (SP)-A on the dynamic surface tension lowering and resistance to inhibition of dispersions of calf lung surfactant extract (CLSE) and mixtures of synthetic phospholipids combined with SP-B,C hydrophobic apoproteins were studied at 37 degrees C and rapid cycling rate (20 cycles/min). Addition of SP-A to CLSE, which already contains SP-B and -C, gave a slight improvement in the time course of surface tension lowering on an oscillating bubble apparatus in the absence of inhibitory protein molecules such as albumin or hemoglobin. However, when these proteins were present at concentrations of 10-50 mg/ml, SP-A substantially improved the resistance of CLSE to their inhibitory effects. The beneficial effect of SP-A required the presence of Ca2+ ions, and disappeared when EDTA was substituted for this divalent cation in the subphase. The effect was also retained when SP-A was heated to 50 degrees C prior to addition to CLSE, but was abolished by heating SP-A to 99 degrees C. Additional studies showed that similar improvements in resistance to inhibition were found when SP-A was added to synthetic mixtures of dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (80:20 by weight) reconstituted with 1% SP-B or SP-B and -C, but not to phospholipid mixtures containing only SP-C. The requirements for SP-B and calcium for the beneficial effects of SP-A on surface activity suggest that the formation of ordered, larger phospholipid-apoprotein aggregates may be involved in the process. The finding that SP-A enhances the ability of CLSE and other surfactant mixtures containing SP-B to resist inhibition is an advantage that will need to be weighed against other factors such as increased antigenicity and heat sensitivity in therapeutic applications in surfactant replacement therapy.

The biophysical activity and inhibition of a series of synthetic surfactant mixtures was studied and correlated with physiological effectiveness in restoring pressure-volume (P-V) mechanics of excised lungs. Results showed that several simple mixtures of dipalmitoyl phosphatidylcholine (DPPC) with fatty acids or diacylglycerols could be formulated to give good adsorption facility and dynamic surface tension lowering to less than 1 mN/m in pulsating bubble measurements at 37 degrees C. However, although biophysical activity approached that of natural lung surfactant (LS) and a related surfactant extract (CLSE) under normal conditions, surface properties were sharply inhibited by relatively small amounts of the plasma protein albumin (2 mg/ml) with minimum surface tensions greater than 30 nM/m even at high surfactant concentrations (5-20 mg lipids/ml). This sensitivity to biophysical inhibition was markedly increased compared to LS and CLSE, and had direct consequences for physiological efficacy: in spite of initially high activity, synthetic surfactants did not exert beneficial effects on P-V mechanics when instilled into surfactant-deficient excised rat lungs. Endogenous protein material was shown to be present upon surfactant recovery by lavage, and bubble measurements confirmed surface activity well below pre-instillation levels. Moreover, full biophysical activity was restored when lavage fluid was extracted to separate the synthetic surfactants from endogenous inhibitors. These results show that it is important to define relative sensitivity to biophysical inhibition in the development of effective lung surfactant substitutes. In addition, the existence of inhibition effects can generate an apparent lack of correspondence between initial biophysical activity and ultimate physiological actions of exogenous surfactant mixtures.

This paper studies the dynamic surface pressure-area (π-A) behavior of phosphatidylglycerol (PG) with a mixed fatty acid distribution (bacterial) in pure and binary mixed films with dipalmitoyl phosphatidylcholine (DPL). At 23°C, bacterial PG films generate maximum dynamic surface pressures of only 48–49 dyn/cm on a 0.15 M sodium chloride subphase for both dilute and surface excess initial conditions. By contrast, binary mixed films of 90:10 DPL/PG reach maximum π values of the order of 70 dyn/cm at similar conditions, the same as for pure DPL films. A collapse plateau ratio criterion is used to show that respreading after dynamic compression past film collapse is enhanced in 90:10 DPL/PG films as compared to pure DPL for a dilute surface initial condition, but not for the surface excess condition, at room temperature. Respreading in pure bacterial PG films is also slightly improved over corresponding pure DPL films for some initial conditions at 23°C, but the magnitude of this effect is not as large as might be expected from the significant unsaturated and cyclopropane fatty acid percentage present in bacterial PG. Differential Scanning Calorimetry (DSC) measurements on DPL/PG mixtures show decreasing T c with peak broadening as the percentage of bacterial PG is increased. The experiments here do not establish a clearly required functional role for 10% PG in pulmonary surfactant surface behavior. Further surface studies are suggested before long-term clinical trials of PG containing mixtures for exogenous replacement therapy in Neonatal Respiratory Distress Syndrome (RDS) are initiated on a widespread basis.

The aerosolization of a multicomponent lipid mixture extracted from bovine lung lavage (surfactant) was investigated in terms of the humidity dependence of the particle size distribution produced by both an ultrasonic and a jet nebulizer. Two methods, sonication on ice and mechanical vortexing, were used to disperse the lipid mixture in 0.15 M NaCl prior to aerosolization in each nebulizer. No significant separation of the lipid from the aqueous phase occurred during nebulization. For both dispersion methods, the particle size dependence on humidity was affected by the presence of dispersed lipids (concentration ∼ 1.5 mg/ml) compared to the case of 0.15 M NaCl aerosols without surfactants. The aerosol from the jet nebulizer consisted of dry particles, and the typical deliquescent behavior for NaCl particles could be demonstrated when relative humidity was increased above 70%. However, when the lipid mixture was nebulized, no such particle growth was observed up to 95% relative humidity. The emerging aerosol from the ultrasonic nebulizer consisted of droplets due to high humidity. In the case of NaCl, these droplets evaporated and became smaller in size with decreasing humidity. For the lipid-saline mixture, the droplets were much more stable and started to evaporate only below 60–70% relative humidity. Although both ultrasonic and jet nebulizers gave particle size distributions in the range for significant alveolar deposition (0.4–1.5 μm AMAD), the ultrasonic nebulizer aerosol sizes at high humidity were optimal in this respect. Calculations of lipid surface and bulk concentration suggest that a significant part of the observed phospholipid dispersion effects on aerosol size behavior may be related to bulk phase interactions.

Surfactant replacement therapy for the respiratory distress syndrome is being actively studied in clinical trials. The therapy has great potential for reducing morbidity and mortality in premature infants. Some issues about surfactant replacement therapy are not yet clear, and a number of these are discussed in this article.

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.

This research studies the biophysical surface activity of synthetic phospholipids combined in vitro with purified lung surfactant apoprotein, having an Mr of 6000. Hydrophobic surfactant-associated protein (SAP-6) was delipidated and purified from both bovine and canine lung lavage, and was combined in vitro with a synthetic phospholipid mixture (SM) of similar composition to natural lung surfactant phospholipids. SM phospholipids were also combined and studied biophysically with another purified surfactant-associated protein, SAP-35. The biophysical activity of synthetic phospholipid-apoprotein combinants was assessed by measurements of adsorption facility and dynamic surface tension lowering ability at 37 degrees C. The SM-SAP-6 combinants had adsorption facility equivalent to natural lung surfactant, and to the surfactant extract preparations CLSE and surfactant-TA used in exogenous surfactant replacement therapy for the neonatal Respiratory Distress Syndrome (RDS). The synthetic phospholipid-SAP-6 combinants also lowered surface tension to less than 1 dyne/cm under dynamic compression in an oscillating bubble apparatus at concentrations as low as 0.5 mg phospholipid/ml. A striking finding was that this excellent dynamic surface activity was preserved as SAP-6 composition was reduced to values as low as 5 micrograms/5 mg SM phospholipid (0.1% SAP-6 protein), an order of magnitude less than the 1% protein content of CLSE and surfactant-TA. Mixtures of SM phospholipids plus SAP-35, the major surfactant glycoprotein, had significantly lower biophysical activity, which did not approach that of a functional lung surfactant. These results suggest that synthetic exogenous surfactants of potential utility for replacement therapy in RDS can be formulated by combining synthetic phospholipids in vitro with specifically purified, hydrophobic surfactant-associated protein, SAP-6.

The time-dependent decay of dynamic surface pressure (π) is investigated for binary films of dipalmitoyl phosphatidylcholine: dioleoyl phosphatidylcholine (DPL:DOL) and of dipalmitoyl phosphatidylcholine:cholesterol (DPL:CHOL) after dynamic compression past monolayer collapse. The results show that the addition of either DOL or CHOL to DPL films acts to increase the degree of relaxation of post collapse surface pressure. Thus, while pure DPL films exhibit a highly stable postcollapse dynamic surface pressure that is more than 20 dyn/cm in excess of the static spread value, films of 50:50 DPL:DOL show dynamic π relaxation to values within a few dynes/centimeter of the static collapse pressure in times of the order of 100 sec at 25°C. Films of 50:50 DPL:CHOL also relax to within a dyne/centimeter of the static spread value at 100 sec, although the actual extent of relaxation is quite small for this specific binary because it is able to generate initial dynamic collapse pressures only a few dynes/centimeter in excess of the static case. Films of both 90:10 DPL:DOL and 90:10 DPL:CHOL show postcollapse dynamic π values with a large extent of relaxation, but the relaxation process is relatively slow and these films show surface pressures still above static spread measurements at the same surface area after 1000 sec. The increased relaxation imparted to DPL films by the addition of DOL or CHOL as a second monolayer component correlates with the effect of these other surfactants in increasing the dynamic respreading in DPL films compressed past collapse on successive cycles of the surface.

This study demonstrates the ability of impacting aerosol droplets containing phospholipids to cause a dynamic film compression state at the air-water interface. The humidity dependence of the lipid aerosol particle size distribution is also determined, including hysteresis effects arising from different directions of humidity change (i.e., humidification vs dehumidification Aerosols were produced by ultrasonic nebulization of surface-active dispersions of dipalmitoyl phosphatidylcholine (DPPC) and egg phosphatidylglycerol (egg-PG) (7:3 molar ratio) in 0.15 M NaCl (1.5 or 10 mg phospholipid/ml Surface activity of aerosols was defined by adsorption measurements and in interfacial deposition studies where a flowing aerosol stream was directed at the surface of a distilled water subphase at 30°C. Continuous deposition of the phospholipid aerosol (linear velocity 150 cm/s) reduced the surface tension of this subphase from 71 dyn/cm to

Adsorption to the air-water interface of natural lung surfactant obtained by bovine lung lavage is compared and contrasted with the adsorption of mixtures of synthetic phospholipids and of extracted mixed lung lipids containing minimal protein. Surface pressure-time (pi-t) adsorption isotherms are measured at 35 degrees C for the surfactant mixtures as a function of the presence or absence of divalent metal cations (Ca2+ and Mg2+) and of heating to 45 degrees C or 90 degrees C. The effect of aqueous dispersion technique (sonication or mechanical vortexing) on the adsorption process is also studied for the extracted or synthetic phospholipid mixtures. The results imply that the protein component is necessary for the optimal adsorption of natural lung surfactant. However, by taking advantage of different methods available for phospholipid dispersion in an aqueous phase in vitro, it is possible to formulate dispersions of extracted lung phospholipids containing of order 1% protein which adsorb as well as the complete surfactant system. These results suggest that protein concentrations in surfactant mixtures can be minimized for applications such as exogenous lung surfactant replacement for the neonatal Respiratory Distress Syndrome (RDS). However, for situations which may involve alterations in endogenous surfactant function such as in lung injury, effects involving pulmonary surfactant protein and protein-lipid interactions may be of functional significance.

Surfactant-associated protein of Mr = 35,000, SAP-35, is the major glycoprotein present in mammalian pulmonary surfactants. In this study, canine SAP-35 and several of its COOH-terminal peptides were purified and characterized by amino acid composition and NH2-terminal sequencing analysis. These proteins were then studied in terms of their specific lipid-binding characteristics and surface activity when combined with a synthetic phospholipid mixture, SM, chosen as an approximation of lung surfactant phospholipids. Purified, delipidated SAP-35 bound SM strongly. In contrast, SAP-21 (a non-collagenous fragment generated by collagenase digestion) bound phospholipid weakly; SAP-18 (an acidic COOH-terminal fragment comprising residues Gly-118 to Phe-231) did not bind phospholipid, demonstrating the importance of hydrophobic amino acid residues Gly-81 to Val-117 and the NH2-terminal collagenous domain in interaction of the SAP-35 with phospholipids. In surface activity experiments, purified SAP-35 enhanced the adsorption of SM phospholipids in terms of both rate and overall surface tension lowering. However, the adsorption facility of the SM-SAP-35 mixture did not approach that of either whole surfactant or the surfactant extract preparations, calf lung surfactant extract or surfactant-TA, used in exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome. In addition, the dynamic surface activity of the SM-SAP-35 mixture was well below that of natural surfactant or surfactant extracts. This was also true of mixtures of SM phospholipids combined with the SAP-18 and SAP-21 fragments of SAP-35.

This article characterizes and analyzes current concepts of the pulmonary surfactant system, and its actions and importance in respiration. Much of the discussion concerns the fundamental role of lung surfactant in the respiratory distress syndrome (RDS) of premature infants, and the success of exogenous surfactant replacement in clinical therapy for this disease. In addition, basic descriptions of the biophysical and physiological activity of lung surfactant are provided to define mechanisms behind surfactant replacement therapy, and how it might be optimized by the further development of appropriate synthetic lung surfactant substitutes.

The interfacial properties of a synthetic liponucleotide [1-β- d -arabinofuranosylcytosine 5′-diphosphate 1,2- l -diacylglycerol (ara-CDPdiacylglycerol)] were investigated at 23°C. Surface properties studied included the quasi-equilibrium surface pressure-area (π-A) behavior of solvent-spread monolayers on a Wilhelmy balance, dynamic surface tension lowering at high cycling rate with an oscillating bubble, and the adsorption kinetics of aqueous dispersions. The π-A isotherm of ara-CDPdiacylglycerol was greatly expanded compared with that of egg phosphatidylcholine (egg PC), which had identical fatty acyl chain composition, and this feature was most pronounced at low surface pressures. Molecular areas of the liponucleotide and egg PC were more similar at higher surface pressures with film collapse areas of 60 and 53 A2/molecule, respectively. Collapse pressures were 40 dynes/cm for ara-CDPdiacylglycerol and 46 dynes/cm for egg PC at a low cycling rate of 4 A2/molecule/min, and these increased only slightly to 44 and 48 dynes/cm, respectively, for a 15-fold increase in cycling rate on the Wilhelmy balance. Maximum surface pressures for these surfactants also did not change significantly in complementary oscillating bubble studies, even at rates as high as 20 cycles/min. Additivity analyses of Wilhelmy balance (π-A) data for mixed monolayers of ara-CDPdiacylglycerol and egg PC suggested that these compounds form ideally miscible films at the air-water interface. Finally, in terms of the additional surface property of adsorption, aqueous dispersions of ara-CDPdiacylglycerol displayed a markedly greater adsorption rate and magnitude than egg PC, correlating with its larger critical micelle concentration. The rapid adsorption found for the synthetic liponucleotide, coupled with its ability to expand phosphatidylcholine monolayers, may give such molecules the potential for biomembrane toxicity.

These experiments characterize the effects of albumin on the dynamic surface activity of natural lung surfactant (LS), and an extracted mixed lipid fraction (CLL), at physiologic temperature, humidity, and film cycling rate on an oscillating bubble apparatus. Measurements of albumin effects on the surface pressure-time (pi-t) adsorption isotherms of CLL and LS are also reported. Results show that albumin in concentrations greater than or equal to 20 mg/ml increased the minimum dynamic surface tension of LS suspensions (0.4 mg phospholipid/ml) from less than 1 dyne/cm to 21 dynes/cm at 37 degrees C. Albumin in low concentrations (2 mg/ml) had a similar detrimental effect on the dynamic surface activity of extracted surfactant lipids, CLL. In addition, albumin also inhibited the isolated adsorption facility of LS and CLL; instead of adsorbing rapidly to their maximum spreading pressures of 45 dynes/cm, both surfactant mixtures (at 0.063 and 0.125 mg phospholipid/ml) adsorbed more slowly or reached lower final surface pressures in the presence of plasma protein. A striking finding was that albumin inhibition of surface activity was moderated or abolished at high lipid concentrations. For example, minimum dynamic surface tensions less than 1 dyne/cm were reached on the oscillating bubble for natural LS at concentrations greater than 0.75 mg/ml and CLL at concentrations greater than 1.5 mg/ml, even in the presence of very large amounts of albumin (100 mg/ml). Similarly, LS and CLL adsorption facility was protected from albumin inhibition at sufficiently high phospholipid concentrations. Albumin inhibition of natural LS adsorption was also moderated by the presence of 1.4 mM Ca2+ ions. These results show that albumin in plasma transudates has the potential to seriously impair alveolar surfactant activity in vivo. However, the detrimental effect will be mitigated if a critical threshold of phospholipid is present.

Measurements of the room-temperature surface pressure-area ( π A) characteristics of dipalmitoyl lecithin (DPL) films reveal that the dynamic π-A compression curve differs markedly from the static compression curve. Since the dynamic compression behavior is essentially independent of cycling rate in the experimental range of 19.2–96A˚2/molecule/min, it appears that the DPL monolayer does not display appreciable viscous effects, although this may not be so over a wider cycling range. Nonetheless, for the experimental range studied, dynamic compression of DPL appears to impart a metastable structure to the surface which is distinct from the static molecular structure. When surface compression is halted at a given film area during a first compression, the DPL surface pressure is found to relax from its dynamic value towards the equilibrium value at the same film area. The relaxation time of this process was only 50 sec at the relatively large molecular area of 68A˚2/molecule, and it increased continuously to a value of 850 sec at an area of 40A˚2/molecule, just prior to film collapse. At all trough areas following film collapse, the surface pressure was exceedingly stable and relaxations of only a few dyn/cm were observed even after 12,000 sec. The data thus indicate that molecular crowding inhibits the relaxation process for DPL films. Moreover, the formation of multilayers during film collapse appears to impart an even greater stability to the film pressure.

Pure component and binary mixed films of the saturated phospholipids dl-α-dipalmitoyl phosphatidylcholine (DPL) and dl-α-dipalmitoyl phosphatidylethanolamine (DPPE) and the plasma proteins fibrinogen and albumin were investigated in terms of dynamic surface pressure-area (π—A) characteristics at 23 and 37°C. The primary conclusions of the studies are: (1) Neither bovine fibrinogen nor albumin significantly alters the maximum surface tension lowering (πmax) achieved by DPL or DPPE at 23 or 37°C. These proteins do, however, expand the π—A isotherms of the saturated phospholipids at low surface pressure and large surface area. (2) Surface initial conditions such as initial surfactant concentration and spreading order can affect the dynamic compression-expansion cycle π—A behavior for binary mixed films of plasma protein-saturated phospholipid, most substantially at large film area. (3) The dynamic surface tension lowering and respreading characteristics of DPL and DPPE films at 37°C and high humidity show correspondence with previously published results at lower temperature in terms of both πmax and nonreproducible hysteresis on successive cycles past collapse. Thus, at physiologically relevant temperatures and humidities, pure DPL lowers water surface tension to

Exposure to high concentrations of oxygen can result in tissue damage, particularly in the lung. Lung pathology induced by hyperoxia includes changes in lung cell populations and morphology. Presumably, alterations in gene expression underlie some of these cellular changes. In order to better understand the molecular basis of these events, a cDNA library was constructed from the mRNA of the lungs of a hyperoxia-exposed rabbit and differentially screened for clones corresponding to hyperoxia-induced messages. This approach has led to the isolation of four clones, three of which are presented in this communication. One clone corresponds to a message whose steady state levels were induced 6-fold and encodes the tissue inhibitor of metalloproteinases, a protein that plays a key role in the regulation of connective tissue turnover in some cells and potentiates erythroid development in others.

The dynamic surface pressure-area properties of pure and binary mixed films of rac-1,2-dipalmitoyl-glycero-3-phosphocholine, 1,2-diocoyl-sn-glycero-3-phosphocholine and cholesterol were investigated at 23 degrees C and 37 degrees C. The pure films and binary mixtures of rac-1,2-dipalmitoyl-glycero-3-phosphocholine: 1,2-dioeoyl-sn-glycero-3-phosphocholine and rac-1,2-dipalmitoyl-glycero-3-phosphocholine:cholesterol were characterized both for dilute surface initial concentrations of 150 Angstrom 2/molecule and for surface excess initial values of 15-50 Angstrom 2/molecule. The results show that the addition of 1,2-dioeoyl-sn-glycero-3-phosphocholine or cholesterol in a binary film with rac-1,2 dipalmitoyl-glycero-3-phosphocholine acts to improve the surface reentry and respreading properties over those of pure rac-1,2-dipalmitoyl-glycero-3-phosphocholine films upon dynamic compression past collapse. This effect is even more pronounced at 37 degrees C than at 23 degrees C, as demonstrated by the application of a collapse plateau ratio criterion. The enhanced dynamic respreading correlates with the effect of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol in decreasing the gel to liquid crystal transition temperature of rac-1,2-dipalmitoyl-glycero-3-phosphocholine water dispersons, as well as with their facilitation of post-collapse dynamic surface pressure relaxation in binary films with rac-1,2-dipalmitoyl-glycero-3-phosphocholine.

Local heat transfer coefficients and fully developed temperature profiles were measured in NaK eutectic mixture in a pipe at uniform wall temperature. Reynolds numbers ranged from 26 000 to 302 000 and flow was fully developed. Consistency of data was affirmed by three independent heat rate measurements. Eddy diffusivity profiles were used to calculate Nusselt numbers in pipes at uniform heat flux. Results for liquid metals are correlated by: Nu(x) = Nu∞1 + 2xDxD > 4 Nuave = Nu∞1 + 8LD + 2LDlnLD4LD > 4 where for uniform wall temperature Nu∞ = 4.8 + 0.0156 Pe0.85Pr0.08, 0.004 < Pr < 0.1 and for uniform wall heat flux Nu∞ = 6.3 + 0.0167 Pe0.85Pr0.08, 0.004 < Pr < 0.1.

An expression for the eddy diffusivity in the turbulent boundary layer near a smooth wall for fluids of high Prandtl number is obtained from emperical considerations: ∈h/v=0⋅00090y+3(1+0⋅0067y+2)1/20 100, fully developed Nusselt numbers calculated with this eddy diffusivity expression are correlated within 4 per cent by the simple equation Nu∞=0⋅0149Re0⋅88Pr1/3

Consideration of heat transfer to a constant property fluid in fully developed turbulent flow in a pipe with a step change in wall temperature leads to a Sturm-Liouville problem. A first order asymptotic technique applied to this problem leads to estimates of heat transfer rates that are in poor agreement with numerical calculations [1]. Presented here is an extension of the first order analysis which yields higher approximations for the lower eigenfunctions. The extension is an unusual application of the method of matched asymptotic expansions in which the expansion parameter is the eigenvalue and matching of both amplitude and phase of functions are required. Analytical expressions are derived but no numerical calculations are given.

The equation describing the turbulent Graetz problem (heat transfer to a fluid in a pipe) is solved numerically for the lower eigenvalues and constants for Reynolds numbers in the range 10 4 Re 6 and for Prandtl numbers in the range 0 Pr 4 . The numerical calculations incorporate new information on eddy diffusivities and are supplemented by asymptotic calculations of the higher eigenvalues and constants of the problem. Heat transfer rates are predicted for both the entry and fully developed regions of the pipe. The numerical results of this study are shown to be in good agreement with experimental data on fully developed heat transfer rates for Prandtl numbers between 0·01 and 10 5 . The numerical predictions are described as follows: Wall thermal condition Correlation Minimum range of validity Uniform temp. Nu ∞ = 4·8 + 0·0156 Pe 0·85 Pr 0·08 ±5% 0·004 Pr 4 Re 6 Uniform flux Nu ∞ = 6·3 + 0·0167 Pe 0·85 Pr 0·08 Uniform flux or temp. Nu ∞ = 5 + 0·016 Re a Pr b a = 0·88−0·24/(4+ Pr ) b = 0·33+0·5 e −0·6 Pr ±10% 0·1 Pr 4 10 4 Re 6 Tables of eigenvalues and constants are provided for Nusselt number calculations in the entry region. Calculations of entry region Nusselt numbers are compared with experimental data, and five per cent heat transfer entry lengths are calculated for both wall temperature boundary conditions. The effect of the wall temperature boundary condition on heat transfer rate is discussed.